Clostridium difficile infection is the most important cause of hospital-acquired diarrhoea. C. difficile is a spore-forming, anaerobic bacterium that is normally present in the intestine of up to 3% of healthy adults and 66% of infants. This organism rarely causes problems in healthy individuals, as it is kept in check by the normal bacterial population of the intestine. However, the use of broad spectrum antibiotics either therapeutically or prophylactically disturbs the balance of bacteria in the gut, allowing resistant C. difficile present as spores to multiply rapidly and produce toxins which cause Clostridium difficile-associated diarrhoea (CDAD—also known as antibiotic-associated diarrhoea), infection of the colonic lumen, pseudomembranous colitis and in extreme cases, death.
There are currently two generally accepted therapies for the treatment of CDAD, namely vancomycin and metronidazole. Both agents are associated with a high (15-20%) rate of recurrence of symptoms, possibly because they do not allow the intestinal flora to return to normal, and agents with an improved profile are greatly needed. In addition, many clinicians are reluctant to prescribe vancomycin long term for fear of increasing general resistance to this antibiotic, which is also used as a “last resort” treatment for other multi-drug resistant pathogens.
Lantibiotics are peptides having antibiotic and other activities, produced by Gram-positive bacteria. They contain, among other modified residues, the thioether amino acids lanthionine and methyllanthionine, which cross-link the peptide chain into a polycyclic structure. They have been classified into two families, type-A and type-B. Type-A lantibiotics are generally elongate amphiphiles that are capable of forming pores in bacterial and other plasma membranes. Examples are nisin and subtilin. Type-B lantibiotics, by contrast, are globular, conformationally defined peptides. Examples are mersacidin and actagardine. Further examples are given in Table 1 below.
The lantibiotics are a class of bacterially produced antibiotic peptides with a narrow spectrum of activity that includes Clostridium difficile. Structurally, the lantibiotics are characterised by extensive post-translational modification primarily involving dehydration of the hydroxyl-containing residues serine and threonine, with optional cyclisations of cysteine onto these dehydrated residues to form thioether bridges.
TABLE 1Classification of LantibioticsType-A LantibioticsType-B LantibioticsNisin AMersacidinNisin ZActagardineSubtilinLacticin 3147EpiderminPlantaricin CGalliderminRuminococcins A and BMutacin BHaloduracinEpicidin 280CinnamycinEpilancin K7DuramycinLactocin SAncoveninLacticin 481PlanosporicinSalivaricin AMichiganin AVariacinAntibiotic 107891
Additionally, Lichenicidin may be classified as a type-B lantibiotic.
Classification according to HG Sahl et al, Ann. Rev. Microbiology 1998, 52, 41 (incorporated by reference herein in its entirety).
Various intermediate forms of lantibiotics also exist and include a number of substances whose biological actions depend on synergy with other peptides. Examples of the latter group include the cytolysins and staphylococcins.
Nisin is the best characterised type-A lantibiotic and has been approved as a food additive in several countries (Turtell and Delves-Broughton, International acceptance of nisin as a food preservative. Bull. Int. Dairy Fed. 1988, 329, 20-23). Nisin is selective for Gram-positive bacteria including Clostridium spp. This narrow spectrum of antibacterial activity would, in principle, make it very well suited as a therapeutic agent for the treatment of CDAD. Unfortunately, nisin is readily metabolised by intestinal enzymes, making its delivery to the site of infection problematic.
A number of attempts to formulate nisin such that it might be delivered to the colon intact have been described (U.S. Pat. No. 5,985,823,1999; T. Ugurlu et al. Eur. J. Pharm. Biopharm. 2007, 67, 202) but the intrinsic vulnerability of nisin to enzymatic cleavage remains a problem.
This has led to the suggestion that nisin and other lantibiotics would need to be encapsulated to target specific areas of the gut to be effective in oral administration (Rea et al. J. Med. Microbiol. 2002, 56, 940). Accordingly, it has been suggested that a bacteriocin should be administered as an enema via an anal route (ibid.).
The present inventors have previously identified and synthesised mersacidin compounds for use as antibiotics (WO 2007/036706). The inventors have also identified and synthesised actagardine compounds, also for use as antibiotics (PCT/GB2007/000138-WO/2007/083112).
The identification of the gene clusters responsible for cinnamycin and duramycin production have been reported, as well as improved methods for their production (see WO 02/088367 and WO 2004/033706 respectively).
Within the field of antibiotics, there is a continuing need for the provision of new antibiotic compounds, to overcome issues such as resistance, bio-compatibility, toxicity and the like. Accordingly, methods of producing lantibiotics, and the production of variant forms of lantibiotics (which may have a different activity profile compared to native forms), are desirable.
For the treatment of a Clostridium difficile infection, it is considered desirable for the antibacterial agent to posses one or more following, amongst others: good in vivo activity against Clostridium difficile; relatively poor activity against most other organisms normally found in the gut flora; little or no toxicity, systemically or in the bowel; and relatively little absorbance when given orally (thereby minimising the systemic effects).
However, until now, there has been a lack of in vivo experimental data to demonstrate the suitability of lantibiotic compounds having antibacterial activity in vitro for use in a method of treatment or prophylaxis of a bacterial infection in a subject. Particularly, there is little data concerning the activity of lantibiotic compounds, and their use in vivo to treat bacterial infections of the colon. The present inventors have now established the in vivo activity of a number of type-B lantibiotic compounds whose use in treatment or prophylaxis has not been previously suggested.